Spiral ganglion neurons (SGNs) and their synapses with the cochlear nucleus (CN) are important components of the auditory system that are impaired in a variety of auditory disorders. Currently, no biological approach exists to regenerate damaged SGNs. Our long-term aims of this proposal are to use stem cell (SC)-derived neurons to replace injured SGNs and regenerate afferent CN synapses that connect the cochlea to the brainstem. In this proposal, we will focus on afferent synapse generation between SC-derived glutamatergic neurons and CN neurons using in vitro and in vivo models. Regenerating the connections between hair cells and SC-derived neurons will be examined in a separate project. A recent study shows that implanted SC-derived cells improved the evoked auditory responses of deafened animals. However, the mechanism (protein-receptor interaction) whereby synapse formation occurs between implanted cells and host CN neurons remains unknown. We have identified neural SCs from mouse cochlear-vestibular ganglia and induced these SCs to differentiate into glutamatergic neurons (ScNs). We observed that neurotrophins stimulated neurite extensions from ScNs. However, neurotrophin supplementation did not significantly promote synaptogenesis between ScNs and CN neurons. Therefore, identification of mechanisms that stimulate auditory synapse regeneration is a major challenge to the restoration of auditory function. Recently, we developed a co-culture model using ScNs and mouse CN neurons to address this issue. We hypothesize that astrocyte-released thrombospondin-1 (TSP1) and the alpha2delta-1 (2-1) receptor are critical for SC-based synaptogenesis. To test this hypothesis, we propose the following specific aims: Aim 1: Determine whether TSP1 is a critical stimulator of ACM-induced synaptogenesis in vitro; Aim 2: Investigate the role of 21 in ACM-induced synaptogenesis in co-cultures; Aim 3: Examine the roles of TSP1 and 21 in CN synapse regeneration in vivo. This proposal studies a protein and a receptor that appear to be critical for the induction of synaptogenesis between SC-derived cells and CN neurons. When our in vitro and in vivo models are optimized in this proposal, we will study the strategies to develop and refine synaptic connections to ensure that the circuit is properly wired in a tonotopic organization in our future research. Identification of proteins and receptors that are important fo CN synapse formation not only will be fundamental to auditory pathway regeneration, but also will provide cues for synapse regeneration in other sensory systems.